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Chapter 4
© 2012 Allahverdiyev et al., licensee InTech. This is an open
access chapter distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro
Adil M. Allahverdiyev, Malahat Bagirova, Serhat Elcicek, Rabia
Cakir Koc, Sezen Canim Ates, Serap Yesilkir Baydar, Serkan Yaman,
Emrah Sefik Abamor and Olga Nehir Oztel
Additional information is available at the end of the
chapter
http://dx.doi.org/10.5772/48403
1. Introduction
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most
common enzymopathological disease in humans. This disease is
described as a widespread, heritable, X-chromosome linked
abnormality (Reclos, et al., 2000). It is estimated that it affects
approximately 400 million people worldwide (Noori-Daloii, et al.,
2004). This disease is seen most frequently in approximately all of
Africa, Asia, and the countries near the Mediterranean Sea (Frank,
2005). G6PD enzyme was demonstrated to play an active role in
survival of erythrocytes. It is known that in the pentose phosphate
pathway of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD)
enzyme provides the production of NADPH and GSH. GSH, produced by
pentose phosphate pathway can react with H2O2 and reduce it to H2O.
This prevents the generation of oxidative stress within red blood
cells; oxidative stress can be induced in erythrocytes whose G6PD
enzymes are deficient. In this situation, GSH is not produced and
H2O2 is not reduced to H2O, leading to oxidative stress and
hemolysis. This is the only mechanism available for the erythrocyte
in order to generate reducing equivalence, therefore making it
essential for the survival of erythrocytes. In individuals whose
G6PD enzyme is deficient, different kinds of hemolysis from mild to
severe are seen bound to differences in variants of the disease
(Beutler, 1983, Luzzatto, 1989).
In epidemiological studies, it was shown that the prevalence of
G6PD deficiency significantly related to malaria. Malaria is known
as a parasitic disease that affects 300-500 million people all over
the world. It is widespread in tropical and subtropical regions of
Asia, Africa and American continents. Five different types of
Plasmodium species—P.
-
Dehydrogenases 66
falciparum, P. vivax, P. ovalae, P. malariae and P.knowlesi—lead
to this disease by infecting erythrocytes. Malaria can become a
life-threatening condition when it is not treated. Each year,
malaria leads to deaths of millions of people all around the world
and a large percentage of deaths are seen in Sub-Saharan regions of
Africa. As it can be easily seen, malaria and G6PD deficiency share
the same geographic distribution. It was shown that G6PD enzyme has
various genetic variants and polymorphic frequencies. Highly
polymorphic frequencies, which are indicators of G6PD deficiency,
are seen in endemic regions for malaria such as Asia, Africa,
Central and South America, while in non-endemic regions, these
rates decrease, suggesting the relationship between G6PD deficiency
and malaria (Haworth, et al., 1988, Organization, 2009, Sutherland,
et al., 2010). This relationship reveals two important results. One
of them is that G6PD deficiency provides great protection from
malaria infection, especially for falciparum infections (Motulsky,
1961, Siniscalco & Bernini, 1961, Ganczakowski, et al., 1995).
On the other hand, G6PD deficiency has been recently demonstrated
to cause serious problems in fighting against malaria. Primaquine,
which is the only drug currently, used in the treatment of
Plasmodium infections leads to severe hemolysis in G6PD-deficient
patients. This drug may even cause death in G6PD-deficient
patients. When primaquine is administered to individuals with G6PD
deficiency, its metabolites lead to more severe hemolysis by
inducing oxyhemoglobin generation, GSH depletion and stimulation of
the hexose monophosphate pathway (Beutler, et al., 1955, Bolchoz,
et al., 2002, Beutler & Duparc, 2007).
Therefore, investigations on detection of G6PD deficiency have a
vital importance for malaria patients before their treatment with
primaquine. On the other hand, the methods that are used for
diagnosing G6PD deficiency are unreliable. Even worse is that it is
very difficult to distinguish heterozygously-deficient patients
from healthy individuals (Peters & Noorden, 2009). All these
data indicate that there is an urgent need to develop new methods
for reliable detection of G6PD deficiency in order to prevent
hemolysis in patients treated with primaquine. Current methods
cannot determine primaquine sensitivity in patients with G6PD
deficiency every time. However, in our previously researches, we
developed a new method for the determination primaquine induced
hemolysis in vitro. This method provides the determination of G6PD
deficiency patients that are susceptible to primaquine
independently from the variants of G6PD deficiency. In our studies,
it was determined that this method demonstrated high sensitivity
for detection of primaquine-induced hemolysis before treatment of
malaria patients with primaquine. This chapter aims to represent
the relationship between G6PD deficiency and malaria, and to
demonstrate the method that has high sensitivity for detection of
primaquine-induced hemolysis in patients with malaria whose G6PD
enzyme is deficient before their treatment with primaquine.
This chapter aims to represent the problems in treatment of
malaria patients with G6PD deficiency by using primaquine,
different methods for determination of G6PD deficiency and a new
method to determine primaquine induced hemolysis before treatment
of patients with G6PD deficiency.
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Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 67
2. Genetic basis of G6PD
G6PD deficiency was identified in 1956 by Carson et al. (Alving,
et al., 1956), and its X-chromosomal inheritance was discerned in
the 1950s by Childs et al. (Childs, et al., 1958). G6PD was cloned
and sequenced by Persico et al.(Persico, et al., 1986, Persico, et
al., 1986) in 1986 and independently by Takizawa and Yoshida
(Takizawa, et al., 1986) G6PD (Misumi, et al., 1982) is in the
hexose monophosphate pathway, the only NADPH-generation process in
mature erythrocytes, which lack the citric acid cycle. Deficiency
of this enzyme in erythrocytes causes various forms of illnesses
such as favism, anemia, chronic nonspherocytic hemolytic anemia,
drug-sensitive hemolytic anemia, primaquine sensitivity and
jaundice in newborns (Beutler, et al., 1968).
By virtue of fact that G6PD is found in all cells, functional
and structural studies have revealed properties of this
housekeeping gene (Luzzatto, 2006). G6PD expression level is
regulated by hormonal and nutritional factors in only a few
tissues. G6PD expression is regulated in liver and adipose tissue,
and its activity depends on the rate of fatty acid biosynthesis
(Greene, 1993). The G6PD gene region is one of the first regions of
the human genome to be completely sequenced (Chen, et al., 1996).
The gene encoding G6PD is located near the telomeric region of the
distal arm of the X chromosome (Pai, et al., 1980, Szabo, et al.,
1984, Patterson, et al., 1987) (band Xq28) and a valuable X-linked
genetic marker for determination of X chromosome inactivations
(Migeon, 1983). G6PD has various polymorphism sites at the G6PD
locus like the colorblindness, Xg blood group and the hemophilia A
locus and has close linkage at the X chromosome (Boyer &
Graham, 1965, Adam, et al., 1967). G6PD is one of a group of genes
including fragile X, (Oberle, et al., 1987) color vision (Motulsky,
1988, Filosa, et al., 1993) hemophilia A (Boyer & Graham, 1965)
clasped-thumb mental retardation syndrome (MASA), (Macias, et al.,
1992) and dyskeratosis congenita (Arngrimsson, et al., 1993)
existing on the distal long arm of the X chromosome.
The X-linkage of the G6PD gene has important implications. This
linkage is very stable and linkage with other group locuses is
similar in all mammals (Luzzatto & Battistuzzi, 1985, Group,
1989, Luzzatto, 1989, Beutler, 1990). In mice, X-linkage of G6PD
was shown by Epstein (Epstein, 1969). Epstein concluded that the
G6PD gene is X-linked in the mouse; its synthesis occurs in the
oocyte and is dosage-dependent. G6PD is a sex-linked and very
polymorphic gene in populations in which males have only one allele
(hemizygous) and females have two G6PD alleles. Thus, females can
be either normal or deficient (homozygous), or intermediate
(heterozygous) phenotypes, whereas males can be either normal or
G6PD-deficient phenotype (Luzzatto, 2006). The frequency of the
deficient phenotype is higher in males than females owing to males
being hemizygous, in which one allele of the gene expresses the
deficient phenotype; to arise in females, G6PD-deficiency needs two
deficient alleles. However, hemizygous deficient males and
homozygous express the same degree of enzyme deficiency level.
Since deactivation of one X-chromosome in embryological development
in heterozygous females have two populations of red cells
(G6PD-normal and G6PD-deficient), with a wide range of total G6PD
enzyme activity depending on the relative proportions. If one of
the alleles contains deficiency, as a result of
-
Dehydrogenases 68
random deactivation of X-chromosomes, about half of the cells
will be normal and the other half will be deficient, although there
is a wide range of variation around that average (Nance, 1964,
Rinaldi, et al., 1976). For this reason, total G6PD activity in
heterozygous females can show variability between near-normal to
near-deficient (Luzzatto & Battistuzzi, 1985, Segel, 2000).
Deactivation of X-chromosome actualizes at random. Correspondingly,
binomial distribution would be expected in deficiency level; the
extent of this distribution depends on X-inactivation time in
embryonic tissue and the number of cells in the embryo.
Furthermore, random deactivation of one X-chromosome engenders
genetic mosaics in heterozygous females (Luzzatto, 2006). As a
result, G6PD mutations show the typical mendelian X-linked
inheritance (Adam, 1961), severe G6PD deficiency is much more
common in males than in females, and X-chromosome inactivation in
heterozygous females for two different G6PD alleles indicate
somatic cell mosaicism (Beutler, et al., 1962, Gall, et al.,
1965).
The total length of the gene is about 18.5 kb on the X
chromosome (xq28) and contains 13 exons. Exon 13 is about 800
nucleotides long and contains the translation stop codon (Nagel
& Roth, 1989, Greene, 1993). The protein-coding region is
divided into 12 segments, ranging in size from 12 to 236 bp
(Martini, et al., 1986). Exon and intron numbers and the exon sizes
and sequences are conserved in higher eukaryotes (Nagel & Roth,
1989, Greene, 1993). The first exon contains no coding sequence and
intron 2 between exons 2 and 3 is extraordinarily long, extending
for 9,857 bp. The function of this long intron is unknown; it may
be important for transcription or processing because compressed
versions of the G6PD gene still have this largest intron in some
species (Chen, et al., 1991, Mason, et al., 1995).
The sequence of the whole G6PD gene is known (Chen, et al.,
1991). G6PD sequence analogy between humans and mice or rats is
87%. The analogy between the mouse and rat cDNA sequences is
greater than humans with 93% similarity. Most of the sequence
dissimilarity is in the 3´- UTR region, which has 600 nucleotides
on average and contains a single polyA site (Nagel & Roth,
1989, Greene, 1993). G6PD gene promoter is embedded in a CpG island
that starts about 680 nucleotides upstream of the transcription
initiation site, extending about 1,050 nucleotides downstream of
the initiation site, and ends at the start of the first intron
(Chen, et al., 1991). CpG island is conserved between some species
(Martini, et al., 1986, Toniolo, et al., 1991), and has highly
enriched guanine and cytosine residues, like characteristically in
other housekeeping genes and this island appears to be preserved
between humans and mice (Toniolo, et al., 1991).
The promoter of the G6PD gene contains a TATA-like, TTAAAT
sequence, and a great number of stimulatory protein 1 (Sp1)
elements (Philippe, et al., 1994, Rank, et al., 1994, Franze, et
al., 1998, Hodge, et al., 1998). These Sp1-binding sites are
essential for promoter activity (Philippe, et al., 1994). Deletion
analysis has uncovered that the “essential” segment of the promoter
is only about 150 bp (Ursini, et al., 1990).
The transcribed region from the initiation site to the poly(A)
addition site covers 15,860 bp. (Chen, et al., 1991). The major
5’-end of mature G6PD mRNA in several cell lines is located
-
Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 69
177 bp upstream of the translation-initiating codon (Martini, et
al., 1986). The G6PD activity and mRNA quantity differ between
tissues (Nagel & Roth, 1989, Greene, 1993). S1 nuclease and
primer extension analyses of mouse G6PD mRNA indicate that when the
transcriptional start site regulated with lipogenesis in liver and
adipose tissue, in kidney G6PD is expressed constitutively (Ho, et
al., 1988); this quantity potentially depends on oxidative stress,
tissue specific differences and reductive biosynthesis reactions
(Nagel & Roth, 1989, Greene, 1993). Some different mRNA forms
of G6PD mRNA have been found, but their functions are completely
unknown. The alternatively spliced form has been documented (Hirono
& Beutler, 1988, Hirono & Beutler, 1989, Cappellini, et
al., 1993), but this mRNA frame contains 138 nucleotides (Mason, et
al., 1988, Persico, et al., 1989, Bautista, et al., 1992, Tang, et
al., 1994). Some researchers (Kanno, et al., 1989) suggested that
in reality, G6PD translation product made from two separate mRNAs
as a result of study to be based on an artifact (Henikoff &
Smith, 1989, Beutler, et al., 1990, Mason, et al., 1990, Yoshida
& Kan, 1990).
Up to 450 G6PD variants have been identified depending on the
enzyme kinetics, physicochemical characteristics, and other
parameters (Luzzatto & Battistuzzi, 1985, Chen, et al., 1991).
Nearly 300 variants of these have been confirmed by the World
Health Organization (1967). Point mutations and small deletions
trigger defects in the enzyme structure. These structural defects
cause altered activity, instability of the enzyme or decreased
affinity of G6PD for its substrates (Luzzatto, 2006).
3. Structure of G6PD and enzymatic properties
G6PD is a typical cytoplasmic, housekeeping enzyme and has been
found in all cells from liver to kidney and organisms, from
prokaryotes to yeasts, to protozoa, to plants and animals (Luzzatto
& Battistuzzi, 1985, Antonenkov, 1989, Glader, 1999, Notaro, et
al., 2000). Inactive form of G6PD is a monomer with 515 amino acids
and has a molecular weight of over 59 kDa (Rattazzi, 1968). The
primary structure of the G6PD enzyme in humans has been determined
from the sequence of full-length cDNA clones (Persico, et al.,
1986). Furthermore, the tertiary structure of the enzyme has been
determined (Au, et al., 1999). Dimer structure of the two subunits
in the enzyme are symmetrically located across a complex interface
of β-sheets (Au, et al., 1999).
Activation of the enzyme requires NADP+ tightly binding to dimer
or tetramer formation of enzyme. G6PD catalyses the first step of
the oxidative pentose phosphate pathway and controls reaction
velocity (Wrigley, et al., 1972). In this first step, while G6PD
catalyses the conversion of glucose 6-phosphate (G6P) to
6-phosphogluconolactone, at the same time it reduces NADP to NADPH
(Au, et al., 1999, Turner, 2000). Human G6PD has no activity with
nicotinamide adenine dinucleotide (NAD) as coenzyme. Also, G6P is
very specific for its substrate compared to other hexose phosphates
(e.g., galactose 6-phosphate or mannose 6-phosphate) (Luzzatto
& Battistuzzi, 1985, Glader, 1999). The G6P binding site is
nearby lysine 205 in tertiary structure of the enzyme, and this
amino acid has a critical role in electron transfer (Bautista, et
al., 1995). The NADP binding site is located nearby 38 to 43 amino
acids; this region constitutes the N-terminus in tertiary structure
encoded in exon 3. This site is important for stability of G6PD
(Au,
-
Dehydrogenases 70
et al., 1999). As an inhibitory effect, one of the products of
G6PD reaction NADPH is an effective inhibitor (Luzzatto, 1967).
Increase in NADP and decrease in NADPH as a result of whichever
oxidative event in cells effect prepotently to increase G6PD
activity (Luzzatto & Testa, 1978). Consequently, G6PD is the
most important enzyme in biosynthesis reactions owing to enzyme
property as NADPH reducer in its critical role in the cytoplasm
(Koehler & Van Noorden, 2003).
4. The effect of G6PD on erythrocyte metabolism
4.1. Erythrocytes
Erythrocytes, which contain hemoglobin, are blood cells that
perform the transfer of oxygen and carbon dioxide between tissues.
G6PD is an important enzyme that performs vital functions within
all cells of the body (Greene, 1993). The quantity of active G6PD
decreases during the life of an erythrocyte and also the older
erythrocytes become vulnerable to oxidative stress. G6PD, an enzyme
in the oxidative pentose phosphate pathway, converts the
nicotinamide adenine dinucleotide phosphate (NADP+) into its
reduced form NADPH. It is necessary for the protection against
oxidative stress in erythrocytes. The cells cannot eliminate this
stress, which causes hemolysis of erythrocytes. Because H2O2 and
other reactive oxygen species cannot be reduced, oxidation of
hemoglobin to methemoglobin and membrane damage occur (Ruwende
& Hill, 1998, Peters & Van Noorden, 2009).
4.2. The importance of pentose phosphate pathway for
erythrocytes
G6PD is the key enzyme in the oxidative pentose phosphate
pathway. The first step of the pentose phosphate pathway is
catalyzed by G6PD. In this step, NADP+ is reduced to NADPH, and
ribulose-5-phosphate, a precursor of DNA, RNA, and ATP, emerge from
G6P (Turner, 2000). The most important reducing agent in the
cytoplasm of cells is NADPH (Koehler & Van Noorden, 2003). The
second enzymatic step in this pathway is NADPH production as a
consequence of reactions that reduce oxidized glutathione (GSSG) to
reduced glutathione (GSH). The only defense against oxidant stress
in the red blood cell (RBC) is GSH production (Friedman, 1979,
Group, 1989, Peters & Van Noorden, 2009). In unstressed, normal
erythrocytes, the G6PD activity is only about 2% of total capacity
(Group, 1989, Peters & Van Noorden, 2009). The pentose
phosphate pathway’s main function is the generation of reducing
capacity through the production of NADPH and ultimately, GSH. This
is essential for cell survival and is available in the erythrocyte
for generating reducing capacity (Greene, 1993).
4.3. Classification of Glucose-6-Phosphate Dehydrogenase
variants
More than 400 variants of G6PD have been distinguished based on
their biochemical characteristics, enzyme kinetics, physicochemical
characteristics, and other parameters (Luzzatto & Battistuzzi,
1985, Chen, et al., 1991, Greene, 1993). G6PD B+ is the most
commonly found enzyme type and it is used as a standard for normal
enzyme activity and electrophoretic mobility. For identification of
other variants, G6PD B+ is used. The rate at
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Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 71
which NADP+ is reduced by glucose-6-phosphate with G6PD B+ as
the catalyst is the standard for activity. Based on this, enzyme
activity relative to G6PD B+ variants are classified as fast,
normal, and slow in terms of electrophoretic mobility and as
Classes I—V (Luzzatto, 1989, Beutler, 1990, Greene, 1993, Segel,
2000, Betke K, Brewer GJ, Kirkman HN, Luzzatto L,Motulsky AC, Ramot
B, and Siniscalco M 1967). There are 5 classes for these variants.
Class I includes chronic nonspherocytic hemolytic anemia with a
severe enzyme deficiency (e.g., G6PD Minnesota, G6PD Tokyo, G6PD
Campinas). Class II variants have severe enzyme deficiency without
chronic nonspherocytic hemolytic anemia (e.g., G6PD mediterrian,
G6PD Canton, G6PD Union, G6PD Kaiping). Class III variants includes
medium or mild enzyme deficiency, with the activity at 10-60% of
G6PD B+ (e.g., G6PD Aˉ). Class IV variants have a weak or no enzyme
deficiency. The activity is 60-100 % of G6PD B+ (e.g., G6PD A+).
Class V variants have increased enzyme activity (e.g., G6PD
Hektoen) (Beutler, 1994, Segel, 2000).
4.3.1. Some Important G6PD Variants
4.3.1.1 G6PD A+ is the most widely seen variant worldwide and
also the first variant in which the nucleotide mutation and amino
acid substitution were determined (Beutler, 1990). This Class IV
variant has 90% of the enzyme activity of G6PD B+ (Luzzatto, 1989).
This variant also called for the African variant cause widely seen
in Africa; 20-40% of African men and 20% of African American men
have this variant. It is faster than G6PD B+ electrophoretically
and it does not cause hemolysis (Beutler, 1989). G6PD A+ derives
from a single amino acid substitution of aspartic acid for
asparagine at amino acid number 126, and this was the result of an
adenine to guanine mutation at nucleotide number 376.
4.3.1.2 G6PD Aˉ is a Class II variant that has 10 to 20% of the
activity of G6PD B+ and the same electrophoretically mobility as
G6PD A+ (Luzzatto, 1989); 11% of African American men have this
variant. Its half-life is 13 days. Three types of mutations have
arisen with molecular studies. The most common mutation being at
nucleotide number 202 is a result of a guanine to adenine mutation
at amino acid number 68 substitution of valine to methionine
(Beutler, 1989, Luzzatto, 1989, Beutler, 1990). The second one
occurs at nucleotide number 680 as a result of a guanine to thymine
mutation at amino acid number 227 substitution of arginine to
leucine. And the third mutation occurs at the nucleotide number
968, as a result of a thymine to cytosine mutation at amino acid
number 323 substitution of leucine to proline (Beutler, 1989). G6PD
A + and G6PD Aˉ variants are defined as unique to Africa, but they
can also be seen in Caucasian populations from Italy, Spain,
Southeast Asia, Middle East and South America (Beutler, 1990).
4.3.1.3 G6PD Mediterranean is a widely seen variant in the
Mediterranean region and Middle East. In addition, it is seen in
the Indian subcontinent and other regions of the Americas (Beutler,
1991). This Class II variant has less than 10% of the enzyme
activity of G6PD B+ and the electrophoretical mobility is similar
with G6PD B+ (Luzzatto, 1989). Its half-life is only 8 days and DNA
analysis identified two different point mutations. The first
mutation is a result of a cytosine to thymine mutation at
nucleotide number 563, at amino
-
Dehydrogenases 72
acid number 188 substitution of serine to phenylalanine
(Vulliamy, et al., 1988). Second is a silent mutation result of a
cytosine to thymine mutation at nucleotide number 1311 (Beutler,
1990). There are many similar Class II variants in the
Mediterranean region (Cagliari, Sassari, El Fayoum), South Asia
(Hong Kong, Canton, Mahidol), and elsewhere. Most of these emerge
as a consequence of point mutations resulting in single amino acid
mutations that have variable effects on activity and
electrophoretic mobility (Luzzatto & Battistuzzi, 1985,
Luzzatto, 1989, Beutler, 1990, Beutler, 1991, Beutler, 1992).
5. Clinical tables on G6PD deficiency
Depending on G6PD enzyme deficiency are: Hemolytic Anemia
(Drug-induced hemolysis), Diabetes mellitus-induced hemolysis and
Infection-induced hemolysis; chronic nonspherocytic anemia, Favism
and Neonatal jaundice.
5.1. Hemolytic anemia
5.1.1 Mechanism of hemolysis. In some people, for example, the
Mediterranean-type, G6PD deficiency from drug intake occurs,
although not a permanent hemolytic condition. In erythrocytes,
NADPH cannot form with G6PD deficiency and unformed NADPH creates a
deficiency in conversion of the oxidized form of glutathione
(GSSG), to its reduced form (GSH) (Lachant, et al., 1984, Beutler,
1994). There is normally plenty of GSH in erythrocytes and it
protects the cell from oxidizing agents. If G6PD is deficient,
hemoglobin is oxidized by oxidative substances to be eliminated and
it returns methemoglobin that cannot function normally. Also,
hemoglobin precipitates with denaturation in the cytoplasm forms
Heinz bodies. These structures attach to the membrane with
disulfide bonds and disrupt its normal structure. The erythrocytes
that contain Heinz bodies in their cytoplasm are sequestered by
macrophages in the spleen and removed from the circulation. G6PD
deficiency hemolysis occurs like that in the extravascular
compartment and also occurs again as a result of membrane defects
(Alving, et al., 1956). Thus, drug-induced hemolysis is the first
and best-known morbid effect of G6PD deficiency. After a 1- or
2-day delay in such drug administration, a fall in the hemoglobin
(Hb) concentration occurs.
The red blood cell (RBC) membrane was adhered to by Heinz
bodies, which are particles of denatured protein. These appear in
the early stages of drug administration and disappear as hemolysis
progresses. Hemolysis usually occurs in blood vessels and
hemoglobinuria follows. The increase of reticulocytes emerges in
response to this situation and the hemoglobin level begins to
increase again within 8-10 days (Beutler, 1994). In severe
hemolysis, the patient may complain of back and stomach pain and
the urine turns dark. The hemolytic anemia is self-limited when
G6PD deficiency is relatively mild because only the older RBCs are
destroyed and the young RBCs have normal or nearly-normal enzyme
activity (Beutler, 1994).
Table 1 lists the drugs and chemicals that cause clinically
significant hemolytic anemia.
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Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 73
5.2. Diabetes mellitus-induced hemolysis. Hemolysis in G6PD
deficiency individuals might initiate diabetic ketoacidosis. This
situation is not exactly accepted. However, hemolysis formation has
been reported when blood glucose levels are normal in diabetic
individuals (Beutler, 1994). It has also has been reported that
hypoglycemia might precipitate hemolysis in patients with G6PD
deficiency (Beutler, 1994).
5.3. Infection-induced hemolysis. Infections are probably the
most common cause of hemolysis in people with G6PD deficiency.
There are numerous reports about the importance of infection in
causing hemolytic anemia. A large number of bacterial, viral and
rickettsial infections have been reported as predisposing factors.
Infectious hepatitis (hepatitis A), pneumonia and typhoid fever are
known to trigger hemolysis. Involving the upper respiratory tract
and gastrointestinal system, viral infections have been reported to
cause a more severe hemolysis (Luzzatto, 2001). The mechanism of
infection-induced hemolysis is not clear, but it is thought to be
that during the infection, superoxide anion and H2O2 production by
macrophages causes the hemolysis (Glader, 1999, Luzzatto,
2001).
5.4. Chronic nonspherocytic anemia
Class I G6PD variants, such as the absence of precipitating
factors in the occurrence of excessive hemolytic anemia, lower
still further the remaining enzyme activity. This is observed in
people with chronic hemolytic anemia and oxidative stress, even if
unstable conditions occur as a result of insufficient enzyme
activity in erythrocytes. Granulocyte dysfunction is seen in some
cases. In these cases, more severe hemolysis is due to increased
susceptibility to infection (Beutler, 1994, Luzzatto, 2001).
5.5. Favism is an illness that occurs in G6PD deficiency
individuals with acute hemolysis by eating raw beans (Vicia fabu).
Wet, dry or frozen fava bean ingestion of grains, even if the
mother eats fava beans can cause hemolysis in newborn infants
through breast milk may occur (Luzzatto, 2001). Individuals with
G6PD deficiency hemolytic effect caused by the beans contained many
glycosides that are toxic due to the visin and konvisin (Beutler,
1994, Akhter, et al., 2011). In addition, β-glucosides in bean
seeds, maturity stage of fava beans attain very high amounts
causing a severe course of hemolytic crisis (Katz & Schall,
1979, Greene, 1993, Beutler, 1994). Often, in the G6PD
Mediterranean variant, acute and a very severe hemolytic crisis are
seen due to fava bean ingestion, even capable of causing death
(Fairbanks, 1999, Luzzatto, 2001). In favism, damage in
erythrocytes is similar to oxidative damage of drugs. Fava beans
include visin, konvisin, ascorbic acid and L-Dopa, which have
oxidative properties. The most commonly cited konvisin and visin
glycosides during digestion fava beans by β-glycosidase or acid
hydrolysis demolished to the active agents, which are converted to
"divisine" and "izouramil." Divisine and izouramilin reduce the
level of the GSH and NADPH in vitro conditions and damage the cell
membrane by the formation of cross-connection with Heinz bodies; it
also has been shown to inhibit Ca2+-ATPase and catalase (Arese
& De Flora, 1990, Beutler, 1994, Gaetani, et al., 1996,
Luzzatto, 2001). 24-48 hours after ingesting foods like fava beans,
characteristic symptoms occur in the form of pallor, jaundice and
hemoglobinuria (Ninfali, et al., 2000). In addition, jaundice,
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Dehydrogenases 74
headache, backache, nausea, fever, and chills are all signs of
acute hemolysis (Tyulina, et al., 2000). Favism is most common seen
in children between the ages of 2-5, and is also 2-3 times more
common in boys than in girls (Luzzatto, 2001). Clinical signs of
favism begin earlier and are more severe than drug-induced
hemolytic crises. Rarely, as a result of pollen of fava inhalation,
hemolysis may occur within hours (Beutler, 1994). While each favism
patient must have G6PD deficiency, hemolytic reactions may not
occur after ingestion of fava beans in each person with G6PD
deficiency. Each individual with G6PD deficiency of the same family
could not be affected in the same way when they eat fava bean. On
the other hand, changes are observed in the same person at
different times. Genetic variations between individuals,
differences of fava bean active metabolites may be responsible for
these variable characteristics (Meloni, et al., 1983, Group, 1989,
Luzzatto, 2001).
5.6. Neonatal jaundice
One of the most threatening consequences of G6PD deficiency is
neonatal jaundice (Beutler, 1994). Jaundice in babies with G6PD
enzyme deficiency could be mild or severe enough to cause
kernicterus, a spastic type of cerebral palsy, and may even cause
death (Luzzatto, 1993). In addition, infants with G6PD deficiency,
hyperbilirubinemia is more remarkable than anemia. It facilitates
this because of the inadequate physiological conjugation in liver
in the neonatal period (Moskaug, et al., 2004). G6PD Aˉ, G6PD
mediterrian, G6PD Canton variants are known as types that cause
kernicterus and hyperbilirubinemia (Luzzatto, 2001). Clinically,
the jaundice, the level of G6PD in the normal physiological
jaundice in newborns occur on the same days, or a little earlier,
but it takes as long as 2-3 weeks (Tan, 1981, Luzzatto, 2001).
There are two major differences between jaundice due to
incompatibility of blood groups and jaundice due to G6PD
deficiency. First, the presence of jaundice in G6PD deficiency is
very rare during childbirth and usually it begins in the second or
third day. Second, according to anemia, jaundice is more pronounced
and it is encountered with severe anemia very rarely in the absence
of the enzyme (Luzzatto, 1993, Luzzatto, 2001).
6. Malaria and glucose-6 phosphate dehydrogenase deficiency
As we mentioned above, there is a strong relationship between
malaria and G6PD deficiency diseases. In several epidemiological
studies, it was shown that distribution of malaria was nearly the
same with distribution of G6PD deficiency (Motulsky, 1961,
Siniscalco & Bernini, 1961, Ganczakowski, et al., 1995). This
situation reveals two important facts. One of them is that G6PD
deficiency provides great protection from malaria, especially for
falciparum infections. On the other hand, using antimalarial drugs
can cause life-threatening hemolytic anemia in patients with G6PD
deficiency. Hence, malaria patients should be screened for their
tendency to G6PD deficiency before their treatment with
antimalarial drugs. In this part, we will first summarize the
importance of malaria for the world. Then, we will explore the
relationship between these two diseases in detail.
As it is known, malaria is a parasitic disease that threatens
300-500 million people all over the world. Malaria can be defined
as the most deadly vector-borne disease in the world
-
Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 75
(Myrvang & Godal, 2000). It is widespread in tropical and
subtropical regions of Asia, Africa and the American continents.
Each year, malaria leads to deaths of millions of people all around
the world and a large percentage of deaths are seen in Sub-Saharan
regions of Africa. The causative agents of malaria are the
Plasmodium parasites, which are transmitted to humans by the bites
of infected mosquitoes. If patients are not treated with
antimalarial drugs, malaria can easily lead to death. Five
different types of Plasmodium species—P. falciparum, P. vivax, P.
ovalae, P. malariae and P.knowlesi—lead to this disease
(Wernsdorfer & McGregor, 1988, Sutherland, et al., 2010).
Plasmodium falciparum (P. falciparum) is the most serious and
life-threatening form of the disease. 80% of death cases are
reported from patients that have been infected with P. falciparum.
It was also demonstrated that resistance has been developed in this
type of parasites against current antimalarial drugs. It is
generally seen in Africa, specifically in sub-Saharan regions.
Interestingly, falciparum-derived malaria cases have been recently
reported in various parts of the world where this parasite species
was believed to be completely eradicated.
Plasmodium vivax (P. vivax) constitutes a milder form of the
disease. Vivax infections generally do not cause death. However,
individuals that suffer from vivax infection also need to be
treated. Among all Plasmodium species, P. vivax is the one that
shows the broadest geographic distribution worldwide. Causative
agents for 60% of malaria infections are reported as P. vivax
infections in India. This parasite has a liver stage and can remain
in the body for years without causing sickness. If the patient is
not treated, the liver stage may re-activate and cause
relapses—malaria attacks—after months, or even years without
symptoms.
Plasmodium ovale (P. ovale) is known as one of the other milder
form of the disease. Like P. vivax, it generally does not commonly
lead to death. Nevertheless, infected individuals require medical
therapy. This parasite, similar to P.vivax, can live in the liver
for long periods without causing symptoms. Therefore, if it is not
treated, reactivation of parasites can be observed in the liver and
this leads to relapse of the disease
Plasmodium malariae (P. malariae) is also another milder form of
the disease. It does not commonly lead to death. However, it still
requires treatment. This type of Plasmodium parasites are reported
to stay in the blood of some individuals for several decades.
Plasmodium knowlesi (P. knowlesi) causes malaria in macaques,
but can also infect humans (Mendis, et al., 2001, Singh, et al.,
2004, Mueller, et al., 2007).
When life cycles of Plasmodium parasites are investigated, it is
seen that the parasites multiply in the liver of the human body,
and then infect erythrocytes. As we mentioned before, Plasmodium
parasites enter the human body when bitten by an infective female
mosquito, which is called Anopheles. These mosquitoes become
infected with malaria when they take Plasmodium-containing blood
from an infected person. Approximately one week later, these
parasites mix with the mosquito's saliva when the mosquito takes
its next blood meal from another person and this individual is
injected with Plasmodium parasites when they are being bitten
(Bozdech, et al., 2003).
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Dehydrogenases 76
Multiplication of the parasites within erythrocytes enhances the
severity of the disease and cause symptoms such as anemia, fever,
chills, nausea, flu-like illness, and, in severe cases, coma, and
death. Treatment of this disease can be achieved by using
antimalarial drugs. Primaquine, which is the most common
antimalarial drug, can be used as a primary prophylactic because it
prevents primary parasitemia of Plasmodium species by destroying
these parasites in the liver before they reach the bloodstream and
cause disease (Yazdani, et al., 2006).
As we pointed out before, according to epidemiological studies,
the prevalence of malaria deeply relates to glucose-6 phosphate
dehydrogenase (G6PD) enzyme deficiency. In these studies, it was
demonstrated that 66 of 77 genetic variants that have reached
polymorphic frequencies were seen in populations living in tropical
and subtropical areas where malaria was endemic. On the other hand,
this genetic diversity does not occur in populations living in
non-endemic regions of the world for malaria, indicating that high
polymorphism is the indicator of G6PD deficiency.
When investigated in terms of cellular biology, we can see that
Plasmodium parasite that causes malaria use erythrocytes as host
cells. Erythrocytes are also the most affected cells from G6PD
deficiency. This situation also suggests the relationship between
the two diseases. In several studies, it was demonstrated that G6PD
deficiency provides a protection against malaria infections. In one
of the early studies, it was indicated that P. falciparum and P.
vivax parasites preferred to invade younger erythrocytes, which
possessed high levels of G6PD enzyme. Since enzyme levels are
diminished in older erythrocytes, parasites do not prefer to invade
these erythrocytes. These studies suggested the protective effect
of G6PD deficiency from parasitemia (Allison & Clyde, 1961,
Kruatrachue, et al., 1962). In the recent past, Ruwando et al. also
carried out a case-control study on more than 2,000 African
children and exhibited that risk of contracting malaria in patients
that have the African form of G6PD deficiency decreased at a rate
of 46 to 58%. In this study, it was suggested that the selective
advantage of resistance to malaria was counterbalanced with
selective disadvantageous results of G6PD deficiency, and this
stopped the rise of malaria frequencies in endemic regions
(Ruwende, et al., 1995). In another study, Ninokata et al. (2006)
investigated 345 healthy adults for G6PD deficiency on Phuket
Island, which had been determined to be a malaria-endemic region
and found out that 10% of these individuals had G6PD deficiency.
Interestingly, it was observed that none of the individuals had
molecular evidence of malaria infection. According to this study,
researchers postulated that G6PD deficiency provided an
advantageous genetic trait against malaria (Ninokata, et al.,
2006).
The exact mechanism of this protection is still unknown. However
there are two postulated explanations. According to the first
suggestion, it was found that parasites that cause malaria can only
survive in conditions with low oxygen levels (Clark, et al., 1989).
This demonstrates that these parasites are very susceptible to
oxidative stress. It is known that in the pentose phosphate pathway
of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD) enzyme
has an important role in production of NADPH and GSH. This is the
only
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Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 77
mechanism for erythrocytes to survive. GSH that is produced by
NADP+ reduction reacts with H2O2 and reduce it to H2O. This
prevents the generation of oxidative stress within red blood cells.
Since oxidative stress is the most important factor for the
disruption of red blood cells, these cells are protected from this
effect. However, in G6PD deficient erythrocytes, G6PD activity is
significantly reduced. In G6PD A (-) variant, enzyme activity level
reduces to 10 or 20% of normal levels, while enzyme activity
completely disappears in G6PD variant. Therefore, oxidative stress
can be induced in erythrocytes whose G6PD enzymes are deficient. In
this situation, GSH is not produced and H2O2 is not reduced to H2O
and leads to oxidative stress. Hence, it is thought that since
malaria parasites are susceptible to oxidative stress, they do not
live within the erythrocytes where their maturation occurs
(Toncheva & Tzoneva, 1985, Greene, 1993). Additionally, during
oxidative stress, the loss of potassium from the cell and from the
parasite can cause the death of the parasite (Friedman &
Trager, 1981).
According to the second suggestion, Plasmodium parasites oxidize
NADPH and reduce the level of reduced glutathione (GSH) in
erythrocytes. In the situation of G6PD deficiency, this effect
becomes more severe and induces oxidative-induced damage within
erythrocytes. Moreover, Plasmodium parasites break down hemoglobin
and release toxic components like iron and these substances lead to
hemolysis. Hence, the development rates of Plasmodium parasites are
diminished. Additionally, red blood cells that are affected by
oxidative stress and are damaged are eliminated by the immune
system via phagocytosis. This elimination decreases the growth of
parasites much more since it occurs during an early ring-stage of
parasites’ maturation. Therefore, all of these data indicate that
G6PD deficiency can provide protection against malaria infections.
Considering the relationship between G6PD deficiency and plasmodium
infections, research has aimed to develop antimalarial drugs that
decrease the level of GSH within erythrocytes and then produce
hydrogen peroxide and the other free radical species in order to
enhance the inhibition of Plasmodium species (Mehta, et al., 2000,
Fortin, et al., 2002, Kwiatkowski, 2005, Prchal & Gregg,
2005).
Primaquine is the only effective antimalarial drug that provides
inhibition of persistent liver stages of P. falciparum, P. vivax,
and P. ovalae parasites that lead to relapses of malaria
(Phompradit, et al., 2011).
However, as we initially mentioned, using primaquine in order to
prevent the relapse of malaria can be very dangerous for G6PD
deficiency patients since its usage results in very severe
hemolysis. In all G6PD variants, activity levels of the enzyme have
been diminished and this partially prevents the defense of
erythrocytes against oxidative attack. However, when primaquine is
administered, its metabolites lead to more severe hemolysis than
oxidative damage by inducing oxyhemoglobin generation, GSH
depletion and stimulation of the hexose monophosphate pathway.
Moreover, primaquine can also induce the generation of Heinz
bodies, which are insoluble aggregates that attach to the surfaces
of erythrocytes. The most probable mechanism of primaquine-induced
hemolysis is the generation of oxyhemoglobin, which forms hydrogen
peroxide. Since G6PD enzyme level is low in G6PD-deficient
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Dehydrogenases 78
erythrocytes, these peroxides accumulate and lead to
denaturation of hemoglobin. Peroxides also generate Heinz bodies
that attach to cell membranes of red blood cells. Hemolysis occurs
when damaged erythrocytes pass through the spleen. In each pass,
red blood cells lose a portion of the cell membrane. After
additional passes, membranes of cells completely lose their
competency (Beutler, et al., 1955, Bolchoz, et al., 2002, Beutler
& Duparc, 2007).
These conditions reach life-threatening scenarios for all G6PD
deficiency patients with different genetic variants. Hence,
individuals that are required to use antimalarial drugs should be
screened very carefully for their tendency to have G6PD deficiency.
For effective control and treatment, either a reliable test for
detecting G6PD deficiency or an anti-malarial drug that can be
safely given to G6PD deficiency patients is required.
7. Detection methods of G6PD deficiency
Currently, primaquine, which causes hemolysis in G6PD-deficient
patients, is the only radical cure of Plasmodium vivax infections
(Burgoine, et al., 2010). Therefore, screening to detect G6PD
deficiency is very important. Various tests can be used for the
detection of G6PD deficiency, which are based on the assessment of
the NADPH production capacity of G6PD. The most frequently used
tests that measure NADPH production are the fluorescent spot test,
cytochemical assay and spectrophotometric assay. However,
fluorescent spot test and the spectrophotometric assay are not
reliable for the detection of heterozygous females. In addition,
DNA analysis can be done to detect G6PD deficiency for the
homozygous, hemizygous, and heterozygous-deficient patients.
However, we have to design primers for all mutations (Peters &
Van Noorden, 2009).
7.1. Fluorescent spot test
Fluorescence is a form of luminescence that uses the physical
change of emission of light upon excitation of molecules. There are
various different types of luminescence, classified depending on
the style of excitation: chemo-luminescence (ending in a chemical
reaction) photo-luminescence (fluorescence, phosphorescence and
delayed fluorescence), bio-luminescence (via a living organism) and
others (Bernard, 2002).
Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is the
reduced form of NADP, with absorption maximum at 340 nm and a
maximum emission at 460 nm. NADPH concentrations have been studied
in great detail using optical methods. A parameter for direct
measurements of the G6PD activity is the fluorescence of NADPH.
When G6PD shows enough functional activity in erythrocytes, two
molecules of NADP+ are reduced to NADPH. After the addition of
glucose 6-phosphate and NADP+, blood spot fluoresces at 340 nm if
NADPH is produced (Beutler & Baluda, 1966).
7.2. Spectrophotometric assay
Spectrophotometric methods are greatly used in biological
sciences for quantitative and qualitative measurements due to the
fact that these methods do not break down the
-
Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 79
molecules analyzed and enable us to assay small quantities of
matter fundamentally (Lehninger, 2000). Spectrophotometric
techniques allow detection of the concentration of a solution by
evaluating its absorbance of a specific wavelength by way of a
spectrophotometer, which produces light at a chosen wavelength and
passes it directly through the sample. Because every molecule have
a specific absorption spectrum, we can recognize and characterize
its properties or detect its current concentration in the presence
of other compounds (Lehninger, 2000).
In the case of enzyme activity measurements, the assay solution
contains some other compounds that are required for the reaction to
occur. Other compounds in the reaction mix may absorb light at the
same wavelength with the enzyme being analyzed. To eliminate the
interference of other compounds, the absorbance of a sample
solution is compared with blank solution, which is taken as the
reference. The blank contains everything found in the sample
solution except the substance to be assayed.
In the matter of protein (enzymatic activity or protein
concentration) measurements, colorimetric methods are used.
Colorimetric measurements are performed by way of quantitative
assessment of a colored complex, which is mostly formed by the
reaction of a colorless compound and a dye reagent. However, the
compound that will be analyzed can be naturally colored and can be
read directly spectrophotometrically.
Glucose-6-phosphate dehydrogenase catalyzes the first step in
the pentose phosphate shunt, oxidizing glucose-6-phosphate (G-6-P)
to 6-phosphogluconate (6-PG). The enzyme activity can be determined
quantitatively by spectrophotometer assay method, which is based on
the rate of NADPH production from NADP+ in G6PD-deficient patients
(Kornberg, et al., 1955, Lohr & Waller, 1974).
These reactions are illustrated below:
Nictotinamide adenine dinucleotide phosphate (NADP) is reduced
by G6PD in the presence of G-6-P. The rate of formation of NADPH is
proportional to the G6PD activity and is measured
spectrophotometrically as in increase in absorbance at 340 nm.
Production of a second molar equivalent of NADPH by erythrocyte
6-phosphogluconate dehydrogenase (6-PGDH) occurs according to the
reaction:
This is prevented by use of maleimide, an inhibitor of
6-PGDH.
The Enzyme Commission of the International Union of Biochemistry
recommends expressing this in international units (IU) and defines
1 IU as the amount of an enzyme that catalyzes the transformation
of 1 micromole of substrate per minute under standard
-
Dehydrogenases 80
conditions of temperature, optimal pH, and optimal substrate
concentration. Specific activity relates activity to total mass of
protein to avoid bias through individual differences in weight
(Bairoch, 1993). Therefore, G6PD activity was expressed as units
(micromoles of NADP reduced per minute) per miligram of soluble
protein at 37°C.
7.3. Cytochemical staining assay
The Cytochemical staining assay is based on the intracellular
reduction of the tetranitro blue tetrazolium (TNBT) by the G6PD via
exogenous electron carrier 1-methoxyphenazine methosulfate and TNBT
is reduced to dark-colored water-insoluble formazan, which can be
determined by light microscopy (Peters & Van Noorden,
2009).
7.4. In vitro primaquine-induced hemolysis methods
3 cc of venous blood anti-coagulated by 2% heparin solution (126
mM NaCl, 14 mM Na2HPO4, 1 mM KH2PO4, 13,2 mM glucose, pH 7.4) was
collected from healthy and G6PD-deficient persons. The blood was
washed three times with sterile heparin solution at 3000 rpm for 10
min. Erythrocytes were resuspended in PBS after that hematocrit was
adjusted to 2%. This is the one of the most important steps for
detection of in vitro primaquine-ınduced hemolysis. Primaquine
solution was prepared in 0.1 M Tris buffer (pH 7.4). Primaquine
concentration was used between 1 and 4 mM in experiments. Different
concentrations of primaquine were added into tubes containing 2%
erythrocytes that were prepared before. Tubes were then placed and
rotated in a rotator tube for 2 hours at 37°C. The rotation speed
was less than 2 rpm. This is another important step for detection
of in vitro primaquine-induced hemolysis. After 2 hours, the
supernatant was collected for the heme concentration, which was
then determined spectrophotometrically. Hemoglobin released from
erythrocyte induction of primaquine-induced hemolysis and compared
with complete lysis (100% hemolysis, control group) obtained by
adding 5 mM Tris-HCl (Fig. 1) (Allakhverdiev & Grinberg,
1981).
The principle of this method is based on conversion of
Hemoglobin (Hb) to cyanmethemoglobin by the addition of KCN and
ferricyanide, whose absorbance is measured spectrophotometrically
as cyanmethemoglobin at 540 nm versus a standard solution.
Supernatant of hemolyzed red blood was diluted four-fold (v/v) with
distilled water. On the other hand, the control group was diluted
twenty-fold (v/v) with distilled water. After that, 50 μL KCN (10%
w/v) and 50 μL potassium ferricyanide (2% w/v) were directly mixed
and the color was measured at 540 nm. The standard curve was
constructed using the standard cyanmethemoglobin solutions in
different concentrations (Bhaskaram, et al., 2003).
This method demonstrated that the in vitro model of
primaquine-induced hemolysis can be only maintained by using 2%
hematocrit in physiological conditions. Primaquine leads to
hemolysis at concentrations between 1 and 4 mM. Other factors that
induce primaquine-derived hemolysis are exposure time, incubation
temperature, drug
-
Glucose-6-Phosphate Dehydrogenase Deficiency and Malaria: A
Method to Detect Primaquine-Induced Hemolysis in vitro 81
Figure 1. In vitro Primaquine-Induced hemolysis
concentration and amount of oxygen. Despite the fact that there
are several methods in order to diagnose G6PD deficiency, these
methods do not determine primaquine sensitivity in patients with
G6PD deficiency every time. Therefore, lack of primaquine-based
treatment by considering only G6PD deficiency can be very dangerous
in terms of health of patients with malaria and the epidemiology of
the disease. On the other hand, treatment of primaquine-sensitivive
individuals with primaquine can cause death. Hence, in the Centers
for Disease Control and Prevention (CDC) report (Hill, et al.,
2006), it was highlighted that there was an urgent need to develop
new in vitro methods for determining hemolysis that indicate
primaquine sensitivity before treatment of patients with this drug.
By considering primaquine-induced hemolysis in patients with G6PD
deficiency, it can be determined whether these patients may be
treated with primaquine or not. The advantage of this method is
that it can determine primaquine-induced hemolysis before treatment
with primaquine and its capacity to determine G6PD deficiency.
8. Conclusion
This chapter has aimed to represent the relationship between
G6PD deficiency and malaria and to suggest a sensitive method for
detection of primaquine-induced hemolysis in patients with G6PD
deficiency. As mentioned above, G6PD deficiency is the most common
enzymopathologic disorder in humans and it affects 400 million
people worldwide. In patients with G6PD deficiency, oxidative
stress cannot be prevented since G6PD enzyme is the initial
catalyst of the pentose phosphate pathway in erythrocytes that
reduces the peroxides to H2O. This situation leads to mild to
severe hemolysis, changing depending on genetic variants of the
disease. As we mentioned before, according to epidemiological
studies, the prevalence of G6PD deficiency deeply relates to
malaria. In these studies, it was demonstrated that 66 of 77
genetic variants, which have reached to polymorphic frequencies,
were seen in populations living in tropical and subtropical places
where malaria was endemic. On the other hand, this genetic
diversity does not occur in populations living in non-endemic
regions of the world for malaria, indicating that high polymorphism
is the indicator of G6PD deficiency and distribution of malaria is
nearly the same with distribution of G6PD deficiency. This
situation reveals two important results.
-
Dehydrogenases 82
One of them is that G6PD deficiency provides partial protection
from malaria infections, especially for falciparum infections. In
several studies, it was demonstrated that risk of contracting
malaria in patients that have G6PD deficiency decreased at a rate
of 46 to 58%. On the other hand, using antimalarial drugs can cause
life-threatening hemolytic anemia in patients with G6PD deficiency.
Since G6PD deficiency does not provide exact protection, these
patients still have a risk of contracting malaria. However, using
primaquine, which is the only radical cure of Plasmodium
infections, can induce more severe hemolysis by generating
oxyhemoglobin, GSH depletion and Heinz bodies and enhancing
oxidative attack. This threatens the lives of patients with G6PD
deficiency. Hence, patients with malaria should be screened for
their tendency to G6PD deficiency before their treatment with
antimalarial drugs. Common methods that are used for diagnosing
G6PD deficiency are unreliable. Even worse is that it is very
difficult to distinguish heterozygously-deficient patients from
healthy individuals. Additionally, current methods cannot
accurately indicate hemolysis, even though they give information
about activity of the enzyme. Also, these methods do not determine
primaquine sensitivity in patients with G6PD deficiency every time.
However, the method that we developed provides the determination of
primaquine sensitivity in patients with G6PD deficiency in vitro
independently from the variants of G6PD deficiency. The principle
of the method is based on the quantitative detection of hemolysis
by incubation of erythrocytes obtained from G6PD-deficient patients
with primaquine in low hematocrit while rotating the culture in a
hybridization oven for 2 hours at 37°C. By considering
primaquine-induced hemolysis in patients with G6PD deficiency, it
can be determined whether these patients may be treated with
primaquine or not. The advantages of this method are that it can
determine the primaquine-sensitivity in patients with G6PD
deficiency before treatment with primaquine. Using this method not
only on G6PD deficiency patients but also on patients that suffer
from other diseases that may cause primaquine-induced hemolysis
constitutes another advantage of the method.
Author details
Adil M. Allahverdiyev, Malahat Bagirova, Rabia Cakir Koc, Sezen
Canim Ates, Serap Yesilkir Baydar, Serkan Yaman, Emrah Sefik Abamor
and Olga Nehir Oztel Department of Bioengineering, Yildiz Technical
University, Istanbul, Turkey
Serhat Elcicek Department of Bioengineering, Yildiz Technical
University, Istanbul, Turkey Department of Bioengineering, Firat
University, Elazig, Turkey
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